Peritubular Contractile Cells in Testis and Epididymis of the Dog , Canis lupus familiaris

Contractile cells surrounding the tubular system of the mammalian testis and epididymis are supposed to contribute to the initial transport of spermatozoa from the testis to epididymis. Testicular peritubular smooth muscle cells have been characterised in detail especially in rodents and humans. The aim of our study was to assess the distribution of peritubular contractile cells of the canine tubuli seminiferi, rete testis channels, ductuli efferentes, and ductus epididymidis by immunohistochemistry and transmission electron microscopy and to classify these cells with respect to their possible physiological function. The entire tubular system of the canine testis and epididymis is surrounded by contractile cells expressing smooth muscle actin, smooth muscle myosin and desmin, which are enveloped, at least partly, by a basal lamina. Some contractile cells of the tubuli seminiferi, rete channels, and efferent ducts and sometimes also single peritubular cells of the ductus epididymidis express vimentin. Contractile cells of seminiferous tubules and efferent ducts represent an intermediate cell type exhibiting characteristics of both smooth muscle cells (SMC) and myofibroblasts, those of rete channels stellate myofibroblasts, and those of the ductus epididymidis SMC. Differences in structure and arrangement of the contractile cells between seminiferous tubules, rete channels, efferent ducts, and ductus epididymidis suggest different functions. Myofibroblasts and contractile cells similar to them could be mainly responsible for the maintenance of an appropriate tissue turgor, whereas contraction of SMC of the ductus epididymidis might cause propulsion of spermatozoa by peristaltic waves. Myofibroblasts, smooth muscle cells, sperm transport, immunohistochemistry, electron microscopy Contractile cells in the peritubular tissue (lamina propria) of the mammalian testis are supposed to contribute to the initial transport of spermatozoa from the testis to the epididymis. In the lamina propria of the seminiferous tubules, contractile cells have been found in many mammalian species such as the rat, mouse, hamster, rabbit, cat, sheep, cattle, pig and human (Roosen-Runge 1951; Ross and Long 1966; Ross 1967; Böck et al. 1972; Bressler and Ross 1972; Bustos-Obregón and Holstein 1973; De Kretser et al. 1975; Hargrove et al. 1977; Wrobel et al. 1979; Maekawa et al. 1996). Similar cells surround the channels of the rete testis (Roosen-Runge and Holstein 1978; Hees et al. 1989). The structure, quantity, and layering of testicular contractile cells vary by species (Maekawa et al. 1996). In early studies, the cells were usually referred to as myoid cells without further classification (Ross and Long 1966; Dierichs and Wrobel 1973; Bustos-Obregón and Courot 1974). Contractile cells surrounding the seminiferous tubules of rodents have recently been analysed in great detail and are usually termed peritubular smooth muscle cells (for overview, see e.g. Maekawa et al. 1996; Tripiciano et al. 1999; Romano et al. 2005, and references therein), although they seem to differ considerably from other smooth muscle cells (Romano et al. 2005). In some species, such as cattle and humans, these cells share characteristics with fibroblasts and have therefore been termed myofibroblasts (Böck et al. 1972; Wrobel et al. 1979; Hees et al. 1989; Holstein et al. 1996; Maekawa et al. 1996). In boars, only cells of the ACTA VET. BRNO 2009, 78: 3–11; doi:10.2754/avb200978010003 Address for correspondence: K. Witter Institute of Histology and Embryology Department of Pathobiology University of Veterinary Medicine Vienna Veterinärplatz 1, 1210 Wien, Austria Phone: +43 1 25077 3403 Fax: +43 1 25077 3490 E-mail: kirsti.witter@vu-wien.ac.at http://www.vfu.cz/acta-vet/actavet.htm inner peritubular layer are contractile, whereas outer layers consist of fibroblasts (Pinart et al. 2001). Studies on contractile cells surrounding rete channels and efferent ducts of mammals are rather rare (Baumgarten et al. 1971; Viotto et al. 1993; Rodríguez et al. 1999; Devkota et al. 2006), although they can also be expected to play a considerable role in the sperm transport. Contractile cells of the lamina propria of the ductus epididymidis are generally characterised as smooth muscle cells (Baumgarten et al. 1971; Whittington et al. 2001; see also textbooks on human and veterinary histology). Myofibroblasts resemble smooth muscle cells (SMC) in their contractility and their cytoskeletal proteins, but fibroblasts in their biosynthetic phenotype and their response to the regulation operated by extracellular matrix and mechanical forces (Lefebvre et al. 1994). They are usually spindle-shaped or stellate with long cytoplasmic extensions and characterised by a discontinuous basal lamina. Compared to SMC, myofibroblasts have a larger Golgi apparatus and abundant rough endoplasmic reticulum, both indicating their synthetic activity (Eyden 2005). The microfilaments are arranged in bundles (stress fibres). Microfilaments form ‘dense bodies’ where they accumulate and insert in plasmalemmal attachment plaques. The nucleus is mostly indented. Immunohistochemical studies have shown that myofibroblasts feature heterogeneous cytoskeletal phenotypes. Hence various types of myofibroblasts according to their content of specific cytoskeletal filaments such as alpha-smooth muscle actin, desmin, myosin and vimentin have been described (JostarndtFögen et al. 1998; Schürch et al. 1998). In contrast, SMC are usually characterised as smooth spindle-shaped cells with numerous membrane caveolae, which are enveloped by a continuous basal lamina. Their cytoplasm contains uniformly distributed microfilament bundles that run in parallel to the long axis of the cells. The nucleus is elongated with blunt ends (Zhang et al. 1997; Schürch et al. 1998; Eyden et al. 1999). However, definition of differentiated SMC is difficult, since variations in marker gene expression depend upon the functional state of the individual cell, as described e.g. for contractile cells of the airways or for vascular SMC (JostarndtFögen et al. 1998; Tang 2008). Precise data on the nature of contractile cells surrounding the tubular system of canine testis and epididymis are not available; the present study was performed to assess the distribution of these cells and to classify them with respect to their possible physiological function. Materials and Methods Samples and preparation of histological sections Testicles including the epididymis of six young adult dogs (Canis lupus familiaris) were obtained after routine orchiectomy requested by the dog owners. The dogs did not suffer from any known impairment of spermatogenesis. Three tissue blocks of a volume of approximately 0.5  2  2 cm were taken from each of these 12 testicles, so that all parts of the tubular system within the organs could be examined. Tissue samples were fixed in buffered formalin according to Lillie, in Methacarn or in Bouin’s solution (Romeis 1989), dehydrated and embedded in paraffin. Sections were cut at 3–5 μm thickness and routinely stained with Mayer’s haematoxylin and eosin. Immunohistochemistry single staining The presence of the cytoskeletal filaments smooth muscle actin, desmin, smooth muscle myosin and vimentin was studied immunohistochemically in order to detect and to classify contractile tissue elements. One section per tissue block and antibody (i.e., 36 sections per antibody) was mounted on poly-L-lysine coated slides, deparaffinised and rehydrated. Endogenous peroxidase activity was blocked with 0.6% H2O2 in methanol, nonspecific binding activity with normal goat serum (DakoCytomation, Glostrup, Denmark) in phosphate-buffered salt solution (PBS) at room temperature. Antigen retrieval by digestion with 0.5 mg protease from Streptomyces griseus (Sigma, Vienna, Austria)/ml PBS for 15 min at room temperature was required for detection of desmin, microwave pre-treatment (2 × 5 min in 0.01 M citrate buffer, pH 6.0) for the detection of vimentin and myosin. Sections were incubated overnight with primary antibodies (manufacturer and dilution, see Table 1) at 4 °C. Products of the immunoreaction of anti-desmin, anti-myosin and anti-vimentin were detected using the anti-mouse PowerVision Kit (Immunovision Technologies, Daly City, CA, USA). Binding of anti-smooth muscle actin was 4

Contractile cells in the peritubular tissue (lamina propria) of the mammalian testis are supposed to contribute to the initial transport of spermatozoa from the testis to the epididymis.In the lamina propria of the seminiferous tubules, contractile cells have been found in many mammalian species such as the rat, mouse, hamster, rabbit, cat, sheep, cattle, pig and human (Roosen-Runge 1951;Ross and Long 1966;Ross 1967;Böck et al. 1972;Bressler and Ross 1972;Bustos-Obregón and Holstein 1973;De Kretser et al. 1975;Hargrove et al. 1977;Wrobel et al. 1979;Maekawa et al. 1996).Similar cells surround the channels of the rete testis (Roosen-Runge and Holstein 1978;Hees et al. 1989).
The structure, quantity, and layering of testicular contractile cells vary by species (Maekawa et al. 1996).In early studies, the cells were usually referred to as myoid cells without further classification (Ross and Long 1966;Dierichs and Wrobel 1973;Bustos-Obregón and Courot 1974).Contractile cells surrounding the seminiferous tubules of rodents have recently been analysed in great detail and are usually termed peritubular smooth muscle cells (for overview, see e.g.Maekawa et al. 1996;Tripiciano et al. 1999;Romano et al. 2005, and references therein), although they seem to differ considerably from other smooth muscle cells (Romano et al. 2005).In some species, such as cattle and humans, these cells share characteristics with fibroblasts and have therefore been termed myofibroblasts (Böck et al. 1972;Wrobel et al. 1979;Hees et al. 1989;Holstein et al. 1996;Maekawa et al. 1996).In boars, only cells of the inner peritubular layer are contractile, whereas outer layers consist of fibroblasts (Pinart et al. 2001).
Studies on contractile cells surrounding rete channels and efferent ducts of mammals are rather rare (Baumgarten et al. 1971;Viotto et al. 1993;Rodríguez et al. 1999;Devkota et al. 2006), although they can also be expected to play a considerable role in the sperm transport.Contractile cells of the lamina propria of the ductus epididymidis are generally characterised as smooth muscle cells (Baumgarten et al. 1971;Whittington et al. 2001; see also textbooks on human and veterinary histology).
Myofibroblasts resemble smooth muscle cells (SMC) in their contractility and their cytoskeletal proteins, but fibroblasts in their biosynthetic phenotype and their response to the regulation operated by extracellular matrix and mechanical forces (Lefebvre et al. 1994).They are usually spindle-shaped or stellate with long cytoplasmic extensions and characterised by a discontinuous basal lamina.Compared to SMC, myofibroblasts have a larger Golgi apparatus and abundant rough endoplasmic reticulum, both indicating their synthetic activity (Eyden 2005).The microfilaments are arranged in bundles (stress fibres).Microfilaments form 'dense bodies' where they accumulate and insert in plasmalemmal attachment plaques.The nucleus is mostly indented.Immunohistochemical studies have shown that myofibroblasts feature heterogeneous cytoskeletal phenotypes.Hence various types of myofibroblasts according to their content of specific cytoskeletal filaments such as alpha-smooth muscle actin, desmin, myosin and vimentin have been described (Jostarndt-Fögen et al. 1998;Schürch et al. 1998).
In contrast, SMC are usually characterised as smooth spindle-shaped cells with numerous membrane caveolae, which are enveloped by a continuous basal lamina.Their cytoplasm contains uniformly distributed microfilament bundles that run in parallel to the long axis of the cells.The nucleus is elongated with blunt ends (Zhang et al. 1997;Schürch et al. 1998;Eyden et al. 1999).However, definition of differentiated SMC is difficult, since variations in marker gene expression depend upon the functional state of the individual cell, as described e.g. for contractile cells of the airways or for vascular SMC (Jostarndt-Fögen et al. 1998;Tang 2008).
Precise data on the nature of contractile cells surrounding the tubular system of canine testis and epididymis are not available; the present study was performed to assess the distribution of these cells and to classify them with respect to their possible physiological function.

Samples and preparation of histological sections
Testicles including the epididymis of six young adult dogs (Canis lupus familiaris) were obtained after routine orchiectomy requested by the dog owners.The dogs did not suffer from any known impairment of spermatogenesis.Three tissue blocks of a volume of approximately 0.5  2  2 cm were taken from each of these 12 testicles, so that all parts of the tubular system within the organs could be examined.Tissue samples were fixed in buffered formalin according to Lillie, in Methacarn or in Bouin's solution (Romeis 1989), dehydrated and embedded in paraffin.Sections were cut at 3-5 µm thickness and routinely stained with Mayer's haematoxylin and eosin.

Immunohistochemistry -single staining
The presence of the cytoskeletal filaments smooth muscle actin, desmin, smooth muscle myosin and vimentin was studied immunohistochemically in order to detect and to classify contractile tissue elements.One section per tissue block and antibody (i.e., 36 sections per antibody) was mounted on poly-L-lysine coated slides, deparaffinised and rehydrated.Endogenous peroxidase activity was blocked with 0.6% H 2 O 2 in methanol, nonspecific binding activity with normal goat serum (DakoCytomation, Glostrup, Denmark) in phosphate-buffered salt solution (PBS) at room temperature.Antigen retrieval by digestion with 0.5 mg protease from Streptomyces griseus (Sigma, Vienna, Austria)/ml PBS for 15 min at room temperature was required for detection of desmin, microwave pre-treatment (2 × 5 min in 0.01 M citrate buffer, pH 6.0) for the detection of vimentin and myosin.Sections were incubated overnight with primary antibodies (manufacturer and dilution, see Table 1) at 4 °C.Products of the immunoreaction of anti-desmin, anti-myosin and anti-vimentin were detected using the anti-mouse PowerVision Kit (Immunovision Technologies, Daly City, CA, USA).Binding of anti-smooth muscle actin was detected with an avidin-biotin-peroxidase-complex (Vectastain ABC Kit™, Vector Laboratories, Burlingame, CA, USA) after incubation of the sections with a biotinylated anti-mouse IgG antibody (Vector Laboratories, Burlingame, CA, USA).The reactions were visualised with diaminobenzidine (Sigma-Aldrich, Vienna, Austria) in 0.03% H 2 O 2 in TRIS buffered saline (pH 7.4).All sections were counterstained with Mayer's haematoxylin, dehydrated and mounted with a medium soluble in xylene.For negative control, normal goat serum was used instead of the primary antibody.

Immunohistochemistry -double staining
In order to characterise the intermediate filaments of peritubular contractile cells in the testis and epididymis, smooth muscle actin/desmin and smooth muscle actin/vimentin double staining was performed.The desmin/ vimentin double staining served for detection of cells with both fibroblastic (vimentin) and smooth muscle (desmin) intermediate filaments.Two sections per staining of four selected methacarn-fixed tissue blocks were prepared (i.e., eight sections per double staining).All sections were deparaffinised and rehydrated as described above.For antigen retrieval, the sections were microwaved for 5 min in Tris-EDTA (DakoCytomation, Glostrup, Denmark; pH 9.0), when necessary.Non-specific binding activity was blocked with normal goat serum (DakoCytomation, Glostrup, Denmark) in phosphate-buffered salt solution (PBS) at room temperature.Afterwards, the sections were incubated with the first antibody (manufacturer and dilution, see Table 1).The immunoreaction was detected with Alexa Fluor 488 goat anti-mouse or Alexa Fluor 568 goat anti-mouse antibodies (Molecular Probes-Invitrogen, Paisley, UK; 1:100).For the second immunostaining, the Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA, USA) was used according to the instructions of the manufacturer, starting with the application of the blocking reagent.The second immunostaining was detected with Alexa Fluor 488 goat anti-mouse or Alexa Fluor 568 goat anti-mouse antibodies as described above, using the colour that was not used for the first staining.

Transmission electron microscopy
Testicles including the epididymis of two young adult dogs were sampled for transmission electron microscopy in such a way that all parts of the tubular system within the organs could be examined.Nine tissue blocks per testicle (i.e.36 specimens) were fixed in 5% phosphate buffered (pH = 7.4) glutaraldehyde and washed in Sörensen's phosphate buffer.The samples were post-fixed in 1% osmium tetroxide in Sörensen's buffer.After a second wash in Sörensen's buffer they were dehydrated and routinely embedded in epoxy resin.Five ultra-thin sections per tissue block were double-stained with lead citrate and uranyl acetate and examined with an EM 900 electron microscope (Zeiss, Oberkochen, Germany).

Results
Contractile cells could be identified in the peritubular connective tissue (lamina propria) in all the examined segments of the male canine reproductive system (Plate I, Figs 1, 2).

Seminiferous tubules
The lamina propria of the seminiferous tubules contained two to three layers of concentrically arranged spindle-shaped cells that stained intensely for actin, myosin and desmin (Fig. 1a-c).Only few cells, predominantly of the middle and outer cell layers contained vimentin (Fig. 1d).In contrast to the vimentin-positive contractile cells in the middle lamina propria layer, peripheral vimentin positive cells were devoid of the smooth muscle markers actin and desmin (Plate II, Fig. 2b-c).
Electron microscopy revealed bundles of collagen fibrils between the cell layers of the tubular lamina propria.The spindle shaped cells which stained positively for the contractile elements were enveloped almost completely by a basal lamina.Nevertheless, some gaps within the basal lamina could be observed.Each cell contained an elongated ovoid nucleus.Cytoplasmic microfilaments were uniformly distributed in the cells, they did not form bundles.Some fibrils coursed parallel to the long axis of the cells.Most fibrils ran, however, more or less transversally avoiding the nucleus and inserting in dense plasmalemmal granular material (attachment plaques).The cell surface was smooth nearly without caveolae.The elongated nucleus had blunt ends and a smooth surface (Plate III, Fig. 3).

Rete testis
The wide channels of the rete testis were lined with a simple cuboidal epithelium partly immunopositive for vimentin or desmin.Their lamina propria contained actin-and myosin-positive cells, which formed an incomplete single layer that was separated from the epithelium by a thick layer of collagen fibres.Single further actin-and myosin-positive cells were situated in the loose connective tissue between the rete channels (Fig. 1f-g).These cells reacted partly for desmin, partly for vimentin (Figs 1h-i, 2d-e).Cells containing both vimentin and desmin were rare (Fig. 2f).
The epithelium was supported by a thick basement membrane.Beneath the basement membrane, dense bundles of collagen fibres as described above and a few elastic fibres intermingled with the contractile cells forming an envelope around the rete tubules.The oblong stellate cells were provided with a discontinuous basal lamina and had several cytoplasmic extensions.Cytoplasmic filaments were not distributed uniformly within the cells.Bundles of filaments in the subplasmalemmal cytoplasm were situated parallel to the long axis of the cell and inserted in prominent plasmalemmal attachment plaques.The cell surface itself appeared smooth without any discernable caveolae.The elongated nucleus featured numerous minor indentations (Plate IV, Fig. 4).

Efferent ducts
Ductuli efferentes were lined by a single layer of columnar epithelium with regular basement membrane.The lamina propria consisted of one to two, sometimes even more, layers of spindle-shaped cells that contained actin, desmin, myosin and vimentin (Fig. 1l-o).Analogical with the contractile cells of rete channels, actin positive cells contained either vimentin or desmin, whereas vimentin/desmin double positive cells were rare (Fig. 2g-i).
All these cells were enveloped by a discontinuous basal lamina.The microfilaments were not distributed uniformly but arranged in bundles (stress fibres) with dense bodies and inserted in plasmalemmal attachment plaques.Numerous caveolae invaginated from the cell surface.The elongated nucleus showed a smooth surface (Plate V, Fig. 5).

Ductus epididymidis
The epithelium of the ductus epididymidis rested on a basement membrane, surrounded by a lamina propria of four and more layers of circularly oriented spindle-shaped cells that showed positive immunoreaction for actin, desmin, myosin and occasionally also for vimentin (Fig. 1q-t).The number of cell layers increased toward the cauda epididymidis without considerable changes in cell morphology or intermediate filament expression.Nearly all contractile cells contained desmin but not vimentin.Vimentin/desmin double positive cells were virtually absent (Fig. 2k-m).
Electron microscopy revealed a continuous basal lamina enveloping these fusiform cells.Microfilaments were abundant, densely packed and distributed evenly within the cytoplasm, parallel to the long axis of the cell.They were associated with numerous dense bodies.Groups of numerous caveolae and interposed attachment plaques were seen at the cell surface (Plate VI, Fig. 6).

Discussion
The entire tubular system of the canine testis and epididymis, including seminiferous tubules, rete channels, efferent ducts and ductus epididymidis, is surrounded by contractile cells, whose morphology and arrangement varies considerably dependent on the segment of the tubular system (Plate VII, Fig. 7).Exact classification of these contractile cells was not always possible, as myofibroblasts are still a controversial type of cells that are difficult to define and identify (Mentzel et al. 1998;Eyden 2001) and also SMC are a heterogeneous cell population depending on the organ, age and functional state (Tang 2008).Most of the testicular and epididymal peritubular contractile cells showed characteristics of myofibroblasts as well as of SMC regarding both immunohistochemical profile and ultrastructure.Strikingly, vimentin immunoreaction was often absent, even if the contractile cells had a distinct myofibroblast ultrastructure.Vimentin/desmin double positive cells could be found in the lamina propria of seminiferous tubules, rete channels, and efferent ducts, but they were rare.In the peritubular tissue layer of ductus epididymidis, they were nearly absent (Fig. 2).
Morphology of contractile cells in the lamina propria of seminiferous tubules suggests that they represent an intermediate between myofibroblasts and SMC.Ovoid nuclei without any discernible indentations, absence of stress fibres, and uniformly distributed microfilaments, which were arranged parallel to the long axis of the cells, are typical for SMC (Schürch et al. 1998).However, the discontinuity of the basal lamina indicates myofibroblasts.Positive immunoreactivity for actin, desmin and myosin does not help to classify these cells definitively, as both SMC and myofibroblasts can express these cytoskeletal proteins.Furthermore, all fibroblastic cells are generally thought to express vimentin (Osborn and Weber 1982).However, in the contractile cells surrounding the canine seminiferous tubules, vimentin positive cells were few and some of them, especially in the outer layer of the lamina propria, could not be classified as contractile cells for their lack of smooth-muscle actin.Similarly as in humans and other larger animals (for review, see Maekawa et al. 1996), the phenotype of canine peritubular contractile cells of the testis seem to show a gradient.The innermost cells adjacent to the basement membrane are more SMC-like, without vimentin but with desmin intermediate filaments, followed by a myofibroblastic cell layer containing even some cells that are staining for vimentin as well as for desmin and an outermost layer of fibroblastic cells.Interestingly, the ultrastructure of cells in the lamina propria cell layers did not differ considerably from each other.
The scattered contractile cells of the rete testis represented typical myofibroblasts (Sánchez et al. 1978;Schürch et al. 1992;Schürch et al. 1998;Powell et al. 1999) with discontinuous basal lamina, cytoplasmic processes, stress fibres inserting in prominent plasmalemmal attachment plaques, few subplasmalemmal caveolae, and nuclei with indentations.However, vimentin/desmin co-expression in these cells could be detected only rarely.
Contractile cells surrounding the efferent ducts showed crucial characteristics of myofibroblasts such as a discontinuous basal membrane, randomly oriented microfilaments, and often positive vimentin staining -either alone or in combination with desmin, but also smooth nuclei and many caveolae as typical for SMC.Just as the contractile cells of the seminiferous tubules they can therefore be classified as an intermediate cell type, but without the phenotype gradient as described above.
The cells building the contractile envelope around the ductus epididymidis were typical SMC with a continuous basal lamina and numerous caveolae.Although the number of cell layers was higher in the cauda epididymidis compared to the caput, the phenotype of contractile cells was constant.
Contractile cells in the mammalian testis supposedly participate in the expulsion of spermatozoa from the testis into the epididymis.Spermatozoa evince little motility while being retained within the testis, and begin to move vigorously only after passage into the epididymis (Hargrove et al. 1977;Trainer 1992).The mechanisms responsible for the initial transport of immotile sperm from the testis into the epididymis are still poorly understood (Middendorff et al. 2002).Several factors have been proposed to influence fluid flow and sperm transport.It was postulated that ciliary action within the efferent ducts moves sperm toward the ductus epididymidis.Recent calculations revealed insufficiency of ciliary action to significantly effect sperm transport (reviewed by Ilio and Hess 1994) but it is still agreed to have some auxiliary function (Wrobel 1998).Constant secretion of fluid by the seminiferous epithelium was found to play a role in the transport of sperm (Ilio and Hess 1994).Another motive source for impelling sperm from the seminiferous tubules through the rete testis into the epididymis, which has been described in various mammals and humans, is the activity of contractile cells of the testicular tunic (Gorgas and Böck 1974;Leeson and Cookson 1974;Leeson 1975;Middendorff et al. 2002), the lamina propria of the seminiferous tubules (Ross and Long 1966;Böck et al. 1972;Langford and Heller 1973;De Kretser et al. 1975;Ellis et al. 1981) or the rete testis (Roosen-Runge and Holstein 1978).In experimental studies, it has been shown directly that these cells do considerably increase sperm transport from the testis (Ilio and Hess 1994).
Differences in structure and arrangement of peritubular contractile cells surrounding seminiferous tubules in comparison with rete channels, efferent ducts and ductus epididymidis, suggest, however, different functions of these cells.Myofibroblasts are known to develop from fibroblasts when loaded by tension (Grinnell and Ho 2002); tension also maintains the myofibroblastic phenotype (Hinz et al. 2001).Increasing substrate stiffness induces increasing force exertion by myofibroblasts (Wrobel et al. 2002).Thus, the main function of myofibroblasts is, additionally to matrix secretion, rather to exert constant force than to dislocate structures (Freyman et al. 2002;Pizzo et al. 2005).In contrast, the primary function of SMC is contraction and relaxation, i.e. shortening and elongation of structures -displacement (Hilgers and Webb 2005).
It can be speculated that myofibroblasts and contractile cells similar to them that surround seminiferous tubules, rete testis channels and efferent ducts, are mainly responsible for the maintenance of an appropriate tissue turgor whithin the testicles, whereas contraction of SMC of the ductus epididymidis might cause true peristaltic movement of this duct and therefore propulsion of spermatozoa.It would be interesting to test whether endothelin, vasopressin, or other substances that are known to induce contraction of peritubular SMC in vitro (for review, see e.g.Maekawa et al. 1996;Palombi et al. 2002;Romano et al. 2005) affect all the different contractile cells as found in the canine testis and epididymis in the same way.In vivo experiments in rats resulting in different changes of luminal pressure in seminiferous tubules compared to the ductus epididymidis (reviewed by Maekawa et al. 1996) showed that this might not be the case.
Fig.1.Immunohistochemical characterisation of peritubular contractile cells in the canine testis and epididymis; tubuli seminiferi (a-e), rete testis channels (f-k), ductuli efferentes (l-p), and ductus epididymidis (q-u).Contractile cells surrounding all parts of the tubular system of both testis and epididymis stain positively (brown) for smooth muscle actin (a, f, l, q) and smooth muscle myosin (b, g, m, r).Desmin positivity is less pronounced in the peritubular cells of rete channels (h) in comparison with the other parts of the tubular system (c, n, s).Most vimentin-positive peritubular cells could be found in the lamina propria of efferent ducts (o; black arrows), whereas in the other parts of the tubular system (d, i, t), vimentinnegative cells (red arrows) prevailed over positive cells (black arrows).Note that epithelial cells lining the rete channels can contain desmin (h, arrowheads) and/or vimentin (i, arrowheads).Control slides, where normal serum was used instead of primary antibodies, showed no positive brown immunostaining (e, k, p, u).Visualisation of the immunoreaction horseradish peroxidase/diaminobenzidine, counterstaining Mayer's haematoxylin.Black arrows -positive immunoreaction, red arrows -negative immunoreaction, arrowheads -positive immunostaining in epithelial cells of rete channels, asterisks -tubules.Scale bar: 50 µm.

Fig. 2 .
Fig. 2. Immunohistochemical detection of intermediate filaments of peritubular contractile cells in the canine testis and epididymis; tubuli seminiferi (a-c), rete testis channels (d-f), ductuli efferentes (g-i), and ductus epididymidis (k-m).In all parts of the tubular system, contractile cells detected by positive actin staining (a-b, d-e, g-h, k-l; green fluorescence) can contain either the muscle-type intermediate filament desmin (a, d, g, k; red fluorescence, overlay yellow) or vimentin (b, e, h, l; red fluorescence, overlay yellow).Desmin and vimentin double positive cells (c, f, i, m; yellow overlay of green vimentin and red desmin fluorescence, arrows) are rare; most of them can be found in the lamina propria of rete testis channels and efferent ducts.Note that epithelial cells lining the rete channels can contain desmin and/or vimentin (arrowheads).Visualisation of the immunoreaction Alexa Fluor 488 goat anti-mouse (green fluorescence) or Alexa Fluor 568 goat anti-mouse antibodies (red fluorescence).Arrows -positive immunoreaction, arrowheads -positive immunostaining in epithelial cells of rete channels, asterisks -tubules.Scale bar: 50 µm.

Fig. 3 .
Fig. 3. Structure of contractile cells surrounding the seminiferous tubules in the canine testis, overview (a) and detail (b).Basal lamina (arrows) with gaps (arrowheads) and attachment plaques (asteriks) are characteristics of myofibroblasts; smooth surface of the nucleus (N), spindle shape and uniform distribution of microfilaments of smooth muscle cells.C -collagen fibres.Transmission electron micrograph.Scale bar: 5 µm.

Table 1 .
Primary antibodies used for immunohistochemistry